Development of porous defects in plasma membranes of adenosine triphosphate-depleted Madin-Darby canine kidney cells and its inhibition by glycine.

Studies during the past decade have led to the recognition of a fundamental, widely expressed mechanism of structural damage in energy-deprived cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+ availability or alterations of cytosolic free Ca2+. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondrial uncoupler in glucose-free medium, and intracellular free Ca2+ was clamped at 100 nM to avoid calcium cytotoxicity. Although the ATP-depleted cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a lipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of graded molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluorescence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules across plasma membranes, with sharp break-points between 70,000 and 145,000 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or "pores," with size-exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the smallest dextran (4,000 d), before enlarging with time to become permeable to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the development of permeability defects. Treatment of cells before ATP depletion with a membrane-impermeant homobifunctional "nearest neighbor" cross-linking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppressed the development of permeability defects, even in the absence of glycine. These observations suggest that the cellular abnormality that is suppressed by glycine involves rearrangement of plasma membrane proteins to form water-filled pores large enough to leak macromolecules.